Mobile swivel and tether device for attachment to laboratory animals

ABSTRACT

A novel swivel tethering system for attaching to a cage of a laboratory animal is disclosed which provides a greater degree of mobility for laboratory animals. This tethering system provides for extended lateral movement which is not limited to the tether length and is ideally suited for chronic indwelling catheterization since it can minimize the stress created in the animals caused by a limited tether length, thereby reducing the potential distortion of blood parameters.

This is a continuation of application Ser. No. 069,839, filed July 6,1987, now U.S. Pat. No. 4,756,707.

FIELD OF THE INVENTION

This invention relates to apparatus for taking experimental data fromunanesthetized laboratory animals, and more particularly, to a swiveltethering system that provide mobility to cannulated, laboratoryanimals.

BACKGROUND OF THE INVENTION

Traditionally, it was common practice to implant arterial or venouscannulas in larger mammals, such as dogs. These animals were then oftenused for repeated administrations of predetermined amounts of drugs.More recently, aorta and right ventricle catheterization of rats andground squirrels has permitted direct measurements of blood pressuresand drug absorption without the errors associated with anesthesia andrestrainment. V. Popovic et al., Permanent Cannulation of Aorta and VenaCava in Rats and Ground Squirrels, J. Appl. Physiol. 15: 727-728, 1960;V. Popovic et al., Technique of Permanent Cannulation of the RightVentricle in Rats and Ground Squirrels. Proc. Soc. Exptl. Biol. Med.113: 599-602, 1963.

Despite these advances, the mouse, a commonly used experimental animal,has been considered too small for permanent catheterization. The smallbody size of these animals has generally limited blood sampling tononsurgical methods. With acute terminal procedures, decapitation andtrunk blood collection or cardiac puncture are often used. Othersituations, however, require multiple sequential blood samples from thesame mouse. The easiest and most common techniques involve collectionfrom the caudal blood vessels in the tail. Following immobilization ofthe mouse, these vessels are either transected, lanced, or punctured toproduce hemorrhage. The other common multiple sampling method isretro-orbital membrane puncture and collection from the ophthalmicvenous plexus. This is generally considered the preferred nonsurgicalapproach for repetitive blood sampling in the mouse. However, thisapproach has drawbacks which include the unavoidable stress of physicalor chemical restraint, the potential for excessive trauma and the humanpsychological discomfort associated with learning and using thistechnique.

One attempt to provide catheterization to unanesthetized mice isdisclosed in P. Popovic et al, Permanent Cannulation of Blood Vessels inMice, J. Appl. Physiol. vol. 25, No. 5, pp. 626, 627 (November, 1968).This article is directed to a technique and apparatus for permanentcannulation for mice using small polyethylene tubings. The cannula ofthis invention is intended to be disposed in a jugular vein or carotidartery for use in cardiovascular studies or the administration of drugsinto the blood stream of the animals. This reference, however, lacks ateaching for a fully mobile catheterization apparatus for reducingstress distortion of blood parameters caused by "leashing" these animalsto a limited circular area. While this short length prevents damage fromchewing, the mouse must be subjected to the stress of almost fullimmobilization whenever the catheter is used.

Another mouse cannula is taught by A. Coquelin et al, Secretion ofLuteinizino Hormone in Male Mice: Factors that Influence Release DuringSexual Encounters, Endocrinology, vol. 106, pp. 1224, 1229 (1980). Thisdiscussion is directed to a study for measuring the sequential changesin blood levels of luteinizing hormone during sexual encounters anddiscloses a chronic cannulation procedure and catheter. The catheteremploys a polyethylene stopper which is sutured to the mouse's back.Also included is a swivel means for supporting the entire unit fromabove and a flare-type tubing adaptor which is soldered to an extensionspring that holds the tubing material of the catheter. Although thisreference discloses a catheterization apparatus for use in smallmammals, such as mice, it also does not disclose a fully mobile devicefor minimizing stress.

Although, not directed to mouse catheter devices, H. Raffe et al,Measurement of Hormones and Blood Gases During Hypoxia in ConsciousCannulated Rats, J. Appl. Physiol.: Respirat. Environ. Exercise Physiol.56 (5), pp. 1426-1430, 1984, teaches apparatus and methods forimplanting chronic arterial cannulas for remote stress-free bloodsampling of conscious unrestrained rats in their own cages. Thereferences discloses the use of lengths of stainless steel spring coilsfor sheathing the cannulas from the rat to the top of the cage. Inaddition to these sheaths, the use of quinidine sulfate is disclosed todiscourage chewing. The cannula of this reference employs polyethylenetubing of various sizes and subcutaneously implanted stainless steeldiscs, having a central hollow stem for receiving part of the springcoil. These discs also include a set screw and four suture holes. Thisreference, however, also fails to disclose a fully mobile in-dwellingcatheterization apparatus suited for mice.

Similarly, A. K. Chatham, Jacket and Swivel and Teathering Systems, LabAnimal, vol. 14 (8), pp. 29-33, 1985, is directed to restraining devicesfor larger animals that may include exteriorized catheters and probesfor infusing drugs and collecting data. The tethering system consists ofa jacket made of a light weight breathable nylon netting material, alight weight, highly flexible stainless steel attaching tether, and acage-mounted swivel to which the tether is anchored. This referencediscloses jacket and swivel tethering systems for use on a variety ofresearch animals, as small as 150 grams, or about five times the weightof an average mouse. As shown in FIG. 3 of this reference, the freedomof movement for the animal is determined by the length of the tether anda fully mobile catheterization apparatus is not disclosed.

Accordingly, there is still a need for a swivel tethering device for usein scientific studies of laboratory animals. There is also a need for afully mobile catheterization apparatus for obtaining serial bloodsamples from nonrestrained and unanesthetized mice without the stressassociated with confinement to a given tether length.

SUMMARY OF THE INVENTION

A swivel tethering system and catheter are provided for attaching to acage of a laboratory animal. This tethering system provides for extendedlateral movement which is not limited to the tether length. The movementof the laboratory animal is facilitated by a track disposed on the topof the cage and a carriage means attached to the tether and disposed onthe track for reciprocal movement along the track. The tethering systemis ideally suited for chronic indwelling catheterization apparatus whichmay be readily disposed coaxially within the tether. Additionally, thisinvention contemplates that a combination of blood-drawing cannulas andelectrical probes could be disposed within the hollow chamber of thetether means.

Thus, a light weight swivel tethering system is provided which overcomesthe stress created in the animals caused by a limited tether length,thereby reducing the potential distortion of blood parameters. It hasbeen observed that adult mice can pull the carriage of this inventionwithout apparent effort.

This invention also provides a chronic indwelling catheterizationapparatus for the collection of multiple blood samples from mice.Uncontaminated and non-hemolyzed samples can be obtained serially fromthe same animal at precise time intervals, while simultaneouslyeliminating potential stress both before and during sample collection.After exiting the mouse, the preferred catheter of this invention can bedirected to the outside of the cage to permit nontraumatic collectionsof multiple uncontaminated blood samples without the necessity ofphysical or chemical restraint. Since at the time of sampling the cageis preferably not open, behavior of the animal rarely changes and themouse generally appears unaware that blood collection is occurring.Flexibility of the wire tether, in conjunction with the fulllongitudinal and rotational mobility of the swivel unit allows a freerange of motion to the mouse while simultaneously protecting thecatheter from being scratched, bitten, or twisted. Materials used inpreparation of the apparatus are readily available and very inexpensive.Pre-surgical assembly of the components of this invention is easy anddoes not require any specialized equipment. Mobile carts, wire tethers,and cage tops can be used repetitively while new catheters are preparedfor each mouse.

In a more preferred embodiment of this invention, a chronic indwellingright atrial catheterization apparatus is disclosed for taking serialblood samples from nonrestrained and unanesthetized mice. Thisparticular system has a light weight carriage means which is generallymade from a washer mounted on four wheels. The tether of this embodimentcan comprise a pipet tip and washer arrangement that provides for freerotation of the catheter. Also disclosed, is a novel means for attachingthe catheter to a mouse which implements a flat perpendicular loopportion of the tether.

It is, therefore, an object of this invention to provide a swiveltethering system that is fully mobile.

It is another object of this invention to provide a swivel tetheringsystem comprising a catheter for obtaining serial blood samples fromnonrestrained and unanesthetized mice.

It is still another object of this invention to provide a swiveltethering system that minimizes potential stress distortion of bloodparameters.

With these and other objects in view, which will become apparent to oneskilled in the art as the description proceeds, this invention residesin the novel construction, combination, arrangement of parts and methodsubstantially as hereinafter described and more particularly defined bythe attached claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate a complete embodiment of inventionaccording to the best mode so far devised for the practical applicationof the principles thereof, and in which:

FIG. 1: is a perspective view of a mouse with a complete catheterapparatus attached;

FIG. 2: is a planar view of the catheter illustrating the protectivesheath and collar;

FIG. 3: is an exploded, planar view of a preferred tether embodimentillustrating a coiled wire, washer and pipet tip.

FIG. 4: is a top planar view of a preferred carriage means for thisinvention.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the teachings of this invention, a swivel tetheringsystem for attaching to a cage of a laboratory animal is disclosed forpermitting a laboratory animal to freely move about its cage. Thetethering system includes a track disposed on a top surface of the cageand a carriage disposed on the track for reciprocal movement thereon. Atether means is provided which is connected to the animal on one end andattached to the carriage at the other. Included in this tethering means,is a conduit means which may include a catheter for taking serial bloodsamples or a electrode for monitoring to the laboratory animal.

Referring now to FIGS. 1-3 which illustrate in perspective, planar andexploded views, a swivel tethering system 100 which is the subject ofthis invention. The swivel tethering system 100 comprises a track 22disposed on a top surface of an animal cage. Disposed on the track 22 isa carriage means that is capable of reciprocal movement along the track22. This invention further includes a tether means, shown in theexploded view of FIG. 3, which is rotatably mounted on the carriagemeans and connected to the animal 10 for tethering the animal 10 to saidcarriage. Finally, a conduit means is disposed within the tether meansfor communicating with the animal 10. As FIG. 1 illustrates, the animal,preferably a mouse, may move freely in its cage, carrying the catheterwithout apparent difficulty. This feature enables experimenters to uselarger cages for permitting a greater degree of movement for the testanimal, while at the same time maintaining a relatively taunt tetherdisposed out of the reach of the animal's teeth.

In a preferred embodiment of this invention, the catheter 40 is disposedwithin said tether means for communicating with said animal 10.Preferably this catheter 40 comprises a protective sheath 44 coaxiallydisposed around a portion of said catheters' length. The catheter 40 canalso comprise a collar 42 disposed near one end of said catheter forserving as a position marker and to stabilize sutures during catheterplacement.

The invention further comprises a tether means, as depicted in FIG. 3,including a coiled wire 32 which can comprise a flat perpendicular loop30 disposed at a bottom portion of said coil 32 for subcutaneousattachment to the laboratory animal 10. The tether means of thisinvention further can include a washer means 34 for rotatable slidingcontact with the carriage means. Further, pursuant to the preferredembodiment of this invention, the tether means can include a pipet tipmeans 12 disposed within an aperture of the washer means 34 for engagingsaid catheter 40. Alternatively, the tether means may comprise anyswivel means of the type generally known to those skilled in the art.

Also included in a preferred embodiment of this invention, is a novelcarriage means which can comprise a wheel for providing reciprocalmovement along said track 22. Preferably this carriage means comprisesat least two axles 50 and 52 disposed in parallel in a body portion ofthe carriage means. As designed, these axles preferably include fourwheels for providing movement to the carriage means. Accordingly to onepreferred embodiment o this invention, the body portion of the carriagemeans is selected to be a washer 20 having an aperture 54 therethrough.Preferably the axles 50 and 52 in this embodiment are disposed on thewasher 20. Finally, accordingly to a most preferred design of thisinvention, the tether means is removably attached to the carriage meansand can be passed into the experimental cage through a slot 24 in thetop of the cage.

The catheter 40 of this invention can be made of any biocompatible,flexible tubing, but preferably consists of a 35.0 cm length of SILASTICtubing by Dow Corning, Midland Mich. Although the length of the tubingcan be varied accordingly to the size of the cage and the experimentalanimal used, a 35.0 cm length has been particularly suitable for use instandard mice cages. The catheter tubing preferably has a inner diameterof about 0.1 to 0.5 mm, more preferably 0.305 mm. The outer diameter ofthe catheter tubing of this invention preferably has a dimension ofabout 0.5 to 0.8 mm, and more preferably 0.635 mm.

In a most preferred embodiment of this invention, two segments ofpolyethylene tubing, obtainable from Clay Adams, Parsippany, N. J., aredisposed over the catheter. A 0.2 cm segment of polyethylene tubing,PE-50 from Clay Adams, is positioned about 0.5 to 3.0 cm, preferably 1.5cm, from the proximal (cardiac) end of the catheter 40. This piece,referred to herein as the "collar" 42, serves as a position marker forthe catheter 40 and also stabilizes sutures during catheter placementand surgery. One method of attaching the collar 42 is to slightlyenlarge the inner diameter of the collar 42 with a hot water bath andneedle forceps to provide easy placement on the silastic catheter 40. Itis important for the purposes of this invention, however, that the fitbetween the collar 42 and the catheter 40 be as snug as possible. Inaddition to the collar, a second tubing segment, also preferably made ofpolyethylene, of about 15.0 to 20.0 cm, preferably 18.0 cm in length, isdisposed over a portion of the length of the catheter 40. Preferablythis second tubing is made from size PE-60 polyethylene tubing from ClayAdams. This segment, referred herein as the "protective sheath",protects the portion of the catheter 40 not beneath the skin surface ofthe animal 10. Finally, the catheter lumen dead space of this inventionis approximately 30 μl.

The tether means of this invention is designed for two distinctfunctions. First, it directs the exposed portion of the catheter 40 upthrough the cage top. Second, it protects the catheter from manipulationor destruction by the mouse. The tether means of this invention ispreferably made of using 26 gauge stainless steel wire, a metal washer(of about 5/16" inner diameter and 3/4" outer diameter), and a 1.0 mlpolyproplylene pipet tip, as substantially described in FIG. 3. Thelength of the tether means depends ultimately upon the depth of the cageand the size of the experimental animal. The tether must comfortablyextend from the dorsum of the experimental animal, preferably a mouse,to the carriage on the top of the cage. The stainless steel wire ispreferably tightly coiled along most of its length with an innerdiameter of approximately 1.5 mm. Since the protective sheath 44preferably has an outer diameter of 1.22 mm, it will fit comfortablywithin the coiled wire 32. Additionally, the top of the coiled wire 32is made progressively wider in order to facilitate attachment to thepolyproplylene pipet tip 12. In this preferred design, the top 1.5 cm ofthe coiled wire 32 is expanded in a funnel shape, accordingly to FIG. 3.It is desired that the exposed coils be very close together in order todeny the mouse any access to the catheter 40. The metal washer 34 isdesigned to be pushed as far as possible onto the pipet tip 12 which isthen attached to the wire coil 32, preferably with ZONAS porous tapefrom Johnson & Johnson Co., New Brunswick, N.J. Finally, the bottomportion of the coiled wire 32 can be formed into a flat perpendicularloop 30 for subcutaneous attachment to the laboratory animal 10.

The preferred carriage means of this invention has the basic design of aminiature cart which includes a metal washer "body", straight pin"axles", and sew-on snap "wheels", as depicted in FIG. 4. Preferably,one size 3, sew-on snap wheel from Dritz Inc. is placed on each of thefour straight pins. Two pins are then taped together to produce an"axle" 50 and 52 . By gluing a front and a back axle to the metal washer20, preferably having dimensions of about 7/16" inner diameter and about1.0" outer diameter, the carriage means is created. It is important tothis embodiment that each wheel 60 spins freely and that the front axlebe disposed in parallel to the back axle so that the carriage meansrolls in a straight line.

The carriage means of this preferred embodiment is designed to supportthe coiled wire 32 which passes into the cage, preferably through abouta 1.2 cm wide median slot in an opaque plastic cage top. Thus, thecatheter 40 can pass up away from the mouse and out the top of the cagecompletely protected within the tether. Blood samples then can becollected without even opening the cage. Experimental and directobservations indicate that catheterized mice are unaware of any changeseven during the actual sampling process. Longitudinal mobility of thecatheter apparatus results as the mobile cart rolls along the cage topon track 24. Rotational mobility is achieved because the tether issupported by but not attached to the carriage means of the preferredembodiment. Again, it has been observed that adult mice can pull thecart and have freedom of movement without apparent effort.

Although surgical methods of catheter placement are known, the followingpreferred procedure proved useful with respect to this mobile system ofthis invention. Phenobarbital was initially used as the anestheticagent. An induction dose of 80 mg/kg body weight (2.4 mg for a 30 gmouse) was then administered intraperitoneally (i.p.) to produce generalanesthesia within 5 minutes. Additional 20 mg/kg i.p. doses were thengiven during the procedure as needed to maintain unconsciousness. Thenails on all hind leg digits were clipped after induction of anesthesia.This practice largely eliminates mouse-inflicted damage to the catheteror suture lines during the period of post-surgical adaptation to thecatheter apparatus. Two surgical sites the right ventral neck and chestregion and dorsally between the shoulders, were next prepared by shavingthe hair and aseptically washing both regions with a dilute mild soapand 70% ethyl alcohol. A stereomicroscope was also used during surgery.The right external jugular vein was then approached through a 1.0 cmventral paramedian skin incision and isolated by careful bluntdissection. The vessel wall is very easy to tear and should never begrasped directly. Two 5-0 silk ligatures were then placed at least 0.2cm apart, both distal to the origin of the axillary vein. The rostralligature was then tightened and a small cut was made in the ventralaspect of the external jugular vein between the two ligatures with irisscissors. The silastic catheter, filled with a heparinized salinesolution (10 I.U./ml)and attached to a microsyringe was then gentlyinserted into the vessel. Difficulty in advancing the catheter may beexperienced at the level of the thoracic inlet. In this event, it ispreferred that the catheter be withdrawn and rotated slightly prior tofurther advancement.

Once the catheter enters the right atria, the beating of the heartagainst the catheter's proximal end will be readily apparent. Thecatheter can then be withdrawn about 2.0 mm to properly position theproximal end just inside the right atria (the beating of the heartshould no longer be felt). Patency of the lumen can be checked bydrawing back on the microsyringe and watching for a free flow of blood.The catheter can be secured by tightening the proximal ligature andtying additional knots around the PE-50 collar. After exiting thejugular vein, the catheter can be directed subcutaneously along the sideof the neck and passed out through a dorsal skin incision between theshoulders. The ventral incision can then be closed.

In a preferred design, an opaque plastic cage top, mobile carriage unit,and wire tether, all supported by a ring stand are positioned over themouse. The catheter 40 can be passed up through the coiled wire 32 andexited through the tether on the outside of the cage top. The tether anbe attached to the mouse by closing the dorsal incision around theperpendicular loop at the base of the coils. The PE-60 tubing sheathpreferably can extend the entire length of the tether in order toprotect the silastic catheter within. Following surgery, the mouse,catheter, tether, and cage top become a single unit and can be movedtogether. The open end of the silastic catheter preferably is occludedwith a 1.5 cm 27 gauge metal brad.

Right atrial catheters of this invention have been successfully placedin mice. Post-surgically, mice have adapted quickly to the presence ofthe catheter apparatus, and within several hours, behavior patterns haveappeared normal. The period of catheter patency has been observed tovary from 10 days to 2 months and can be improved as surgical andcatheter management experience is accumulated. On the average, however,experimental use of the preferred catheters of this invention indicatedthat these devices remained fully patent for 21.5 days allowing morethan 4.0 ml of blood to be obtained per mouse. The experimentalcollection schedule, 250 μl of blood collected for 3 successive daysfollowed by 1 day of rest, resulted in a temporary red blood cellanemia. The average hematocrit dropped by to 31% by day 3, but returnedto normal limits by day 5. Peripheral reticulocytes, counted to monitorthe hematopoietic response, rose to a corrected value of 14% by day 6and stabilized at approximately 7% by day 11. Moreover, total plasmaprotein values did not change significantly. Corticosteroneconcentrations in blood samples obtained from catheterized mice weresimilar to levels in rapidly killed nonstressed controls. However,stress in the form of physical handling for a duration of 15 minutesprior to trunk blood collection markedly elevated plasma corticosteroneconcentrations in both sexes, with a greater response in females.

From the foregoing, it can be realized that this invention provides animproved swivel tethering system for attaching to a cage of a laboratoryanimal. Specifically a fully mobile chronic indwelling right atrialcatheterization apparatus has been disclosed in order to obtain serialblood samples from nonrestrained and unanesthetized mice. The system isunique in that, despite the small size of the mice, multiple,uncontaminated blood samples can be obtained over extended periods oftime while the animals remain completely undisturbed. Potential stressdistortion of blood parameters due to handling, restraint and leashingthe animal to a limited circular area, therefore, is effectivelyeliminated. The apparatus has special application to blood clearancestudies and for the determination of changes in the blood concentrationof endogenous compounds over time. As disclosed, the preferred catheterapparatus comprises three parts: the catheter, a stainless steel wiretether means, and a carriage means. Each of these parts can be preparedbeforehand and easily assembled during surgical placement of thepreferred catheter in a mouse. Although various embodiments have beenillustrated, this was for the purpose of describing, but not limitingthe invention. Various modifications, which will become apparent to oneskilled in the art, are within the scope of this invention described inthe attached claims.

I claim:
 1. A swivel tethering system for attaching to a cage of alaboratory animal, comprising:(a) a track disposed on a top surface ofsaid cage; (b) a carriage means disposed on said track for extendedlateral movement along said track; (c) a tether means rotatably mountedon said carriage and connected to said animal for tethering said animalto said carriage; and (d) conduit means disposed within said tethermeans for communicating with said animal.
 2. The system of claim 1wherein said conduit means comprise a catheter.
 3. The system of claim 1wherein said conduit means comprises an electrode.
 4. The system ofclaim 2 wherein said catheter comprises a protective sheath coaxiallydisposed around a portion of said catheter's length.
 5. The system ofclaim 4 wherein said catheter further comprises a collar disposed nearone end of said catheter.
 6. The system of claim 1 wherein said tethermeans comprises a coiled wire.
 7. The system of claim 1 wherein saidtether means further includes a washer means for rotatable slidingcontact with said carriage means.
 8. The system of claim 7 wherein saidtether means further includes a pipet tip means disposed within anaperture of said washer means for engaging said catheter.
 9. The systemof claim 1 wherein said tether means comprises a swivel.
 10. The systemof claim 9 wherein said carriage means comprises a wheel for providingreciprocal movement along said track.
 11. The system of claim 1 whereinsaid carriage means comprises at least two axles disposed in parallel ona body portion of said carriage means, said carriage means having atleast four wheels attached to said axles.
 12. The system of claim 11wherein said body portion comprises a washer.
 13. The system of claim 12wherein said axles are disposed on said washer.
 14. The system of claim1 wherein said tether means is removably attached to said carriagemeans.
 15. The system of claim 14 wherein said tether means is passedinto said cage through a slot in the top of said cage.
 16. The system ofclaim 6 wherein said coiled wire comprises a flat perpendicular loopdisposed at a bottom portion of said coil for subcutaneous attachment tosaid laboratory animal.